The overall objective is to use established methodologies to determine the intra-nuclear distribution of rat uterine receptors throughout the period during which receptors translocate to the nucleus and the period during which receptors are processed. Translocation and processing are divided into temporal stages, in each of which chromatin is fractionated. This approach should identify chromatin components which dynamically interact with receptors and which will be important for defining the mechanisms of receptor action. The perspective of this proposal recognizes the substantial potential for redistribution by chromatin components during chromatin preparation, disruption and fractionation. Methodologies are presented for maximizing the identification of genuine receptor-chromatin interations by employing multiple fractionation strategies which very rapidly disrupt chromatin with nucleases. This proposal describes the use of 1) purified nuclei from homogenized tissue in order to examine receptor distribution in fractionated chromatin 2) nuclei obtained after Triton-X-100 mediated cell lysis in order to reduce time-dependent redistributions which occur during homogenization and subsequent preparation and 3) chemical cross-linking in order to stabilize receptor-chromatin interactions in situ. The research objectives of this proposal are 1) to characterize the dynamics of the intranuclear distribution of estrogen receptors by performing chromatin fractionation throughout the period of receptor translocation, 2) to identify chromatin fractions which represent the sites of estrogen receptor processing and 3) to explore the use of proteases to subfractionate chromatin and to further resolve the intranuclear localization of estrogen receptors. The unique feature of this proposal is that, in addition to its attempt to resolve chromatin components which interact with receptors in a model system, it develops an approach which will have direct application for analyzing the distribution of steroid receptors in the chromatin of steroid-responsive human cancers, where processes leading to malignancy may have introduced a nuclear alteration which interacts with the steroid receptors of the transformed target tissue in such a way that abnormal development results.